The long term objective of this project is two-fold. First, our aim is to study cell-to-cell communication in the taste bud. One of our Specific Aims is to investigate which neurotransmitters are involved at synapses in taste organs. We plan to use a battery of neurochemical and molecular biological techniques, including immunocytochemistry, quantitative autoradiography, high pressure liquid chromatography with electrochemical detection, and polymerase chain reactions to investigate transmitters. We will determine which transmitters are present in taste buds; which ones are taken up by taste cells and released by calcium-dependent mechanisms when the cells are stimulated; where the transmitters are localized; which transmitter receptors are expressed in taste cells; and where these postsynaptic receptors are expressed. A major focus of the studies will be on the possible neurotransmitter role of biogenic amines and on amino acid transmitters, particularly gamma aminobutyric acid (GABA) and glutamate. Other transmitter and neuromodulator candidates, especially neuropeptides, will also be investigated. We will also apply patch clamp recording techniques in a new preparation, the lingual slice, to study electrical synapses between taste cells. We will use a novel method for monitoring electrical connections in the living isolated taste bud and test whether taste stimulation (e.g. acids) change the properties of the electrical connections. We will also test whether neuromodulators such as serotonin affect electrical coupling between taste cells. Second, we will use the new lingual slice preparation to study how chemical stimuli affect the apical, chemosensory surface of taste receptor cells. Our Specific Aim here is to determine whether taste cells are "tuned" to one or only a few chemical stimuli or whether taste cells respond broadly to a number of different, unrelated chemical stimuli. We will use patch recording techniques to record receptor current evoked by focal application of chemical stimuli onto the apical pore of taste buds isolated in the lingual slice preparation. The project will focus on how two specific classes of chemical stimuli, acids and calcium salts, affect taste receptor cells. These are both important taste stimuli in man and animals. Acids generally taste sour and calcium salts often are bitter. We will test several hypotheses that have been put forth to explain transduction of these chemicals, such as decreases in apical potassium conductance, influx of protons, involvement of ion transporters. We will patch clamp taste receptor cells and analyze membrane conductance changes, if any, elicited by these chemical stimuli. In the broad view, our experiments may lead to the rational design of pharmaceutical agents that can suppress, enhance or modify taste since we will determine what neurotransmitters are acting in the taste organs and since our data will lead to an understanding of how chemicals act on the apical, chemosensory membrane of taste cells. Our study will also bear on all chemosensitive cells, generally, such as pH receptors, wherever they occur in the central and peripheral nervous system.